In order to diagnose various diseases and examine progression of disease conditions, a practice of measuring a content of protein which exists in human body fluid and is characteristic to each disease has been in wide use in the medical field.
For measurement of contents of such proteins, typically, immunoreaction measurement methods utilizing highly specific antigen-antibody reactions have been broadly used, and at present, immunoreaction measurement methods with various principles applied thereto have been developed and utilized.
Among them, measurement methods for detecting an agglutination complex generated by an antigen-antibody reaction, such as nephelometry, turbidimetry and slide agglutination, are well known. These methods are performed using a solution in which an antigen and antibody are uniformly dispersed, and therefore, are collectively called homogeneous immunoreaction measurement methods.
These reactions lead to generation of agglutination complexes, and a reaction solution becomes turbid depending on amounts of an antigen and antibody. Nephelometry and turbidimetry are methods of optically measuring the turbidity, and in nephelometry, turbidity is measured based on an amount of light scattered by a reaction system; in turbidimetry, turbidity is measured based on an amount of transmitted light reduced by scattering in a reaction system. In general, the same reaction solution (reaction system) can be used as subject substances of both the methods, and a subject which can be measured by the one method can also be measured by the other method.
Slide agglutination is a method for determining turbidity caused by generated agglutination complexes by visual observation on a slide glass or the like, and can employ the same reaction system as used for nephelometry and turbidimetry.
In the above-described conventional homogeneous immunoreaction measurement methods, various additives have been tested for accelerating an antigen-antibody reaction to measure a trace amount of component with high sensitivity. As a well-known example mentioned can be a method of improving reaction time and measurement sensitivity by allowing a water-soluble polymer, such as polyethylene glycol (PEG), dextran, polyvinylpyrrolidone or polyvinyl chloride, to coexist in a reaction system so as to accelerate formation of agglutination complexes due to an antigen-antibody reaction.
Among these water-soluble polymers, polyethylene glycol is known to have a high level of effect even at a relatively low concentration, and polyethylene glycol having an average molecular weight of 6000 has been widely used at a concentration of 2 to 6 wt %. Particularly, a 4 wt % concentration is believed to produce only a small level of non-specific turbidity, and thus highly effective.
The effect of accelerating an antigen-antibody reaction by a water-soluble polymer typically has a tendency to be large, as the molecular weight, or the concentration of the polymer in an aqueous solution to be used, is high. Concerning measurement of an antigen-antibody reaction, the higher the degree of an antigen-antibody reaction, i.e. the intensity of a signal depending on the concentration of an antigen, the more favorably the S/N ratio can be kept and the more stably the measurement can be performed. However, when an attempt is made to obtain the above-described effect by further acceleration of an antigen-antibody reaction, in the case of adding a conventional water-soluble polymer, a water-soluble polymer with a higher concentration or a higher molecular weight is required. This however leads to an increase in viscosity of a solution, in which such a water-soluble polymer is dissolved, raising a problem in that the solution is difficult to handle during manipulation for analysis.
In homogeneous immunoreaction measurement methods, a phenomenon called the zone phenomenon is generally known. The zone phenomenon refers to a phenomenon that, when an amount of one of an antigen and antibody exceeds the equivalent weight region thereof which forms the largest amount of agglutination complexes, generation of the agglutination complexs becomes difficult. A binding reaction between a polyvalent antibody and a more than monovalent antigen is explained by the famous Heidelberger's lattice hypothesis, the details of which are described in Fundamental Immunology, William E. Paul, (1984) (Japanese language translation, Kiso Menekigaku, supervised by Tomio Tada, pp. 714–716 (1987)).
In actual homogeneous immunoreaction measurements, an antibody is often used to measure an antigen concentration. A measurement value often has a more important meaning when the antigen concentration is high than when it is low. Therefore, a zone phenomenon due to excess antigens may often cause problems. In regions other than a prozone, a huge molecular chain comprising a complex, in which antibodies and antigens are alternately linked together, is generated, and the amount or size of the molecular chain is increased depending on the antigen concentration if the antibody concentration is constant. Measurement of the amount or size of the molecular chain as optical variations allows quantitative determination of the antigen concentration. Further, since an antigen-antibody complex can be sufficiently observed even by naked eye as turbidity or agglutination s in a solution, depending on the concentrations of an antibody and antigen, the antigen concentration can be qualitatively determined by visual observation or the like.
However, in an antigen excess region, the presence of an antigen in an excessively larger amount than that of an antibody causes an increase in amount of the antibody whose binding site is saturated with the antigen. For this reason, generation of a molecular chain as described above becomes difficult, and the reaction result in this case cannot be readily distinguished from the reaction result in the case of a low antigen concentration. Accordingly, correct quantification and determination depending on the antigen concentration cannot be performed, raising a problem in that a concentration range to be measured has to be limited in order to avoid such a situation.
As measures for improving this zone phenomenon, the following measures have been proposed.
For example, in Japanese Laid-Open Patent Publication No. Hei 09-08984, a method is disclosed in which the concentration of sodium chloride is set to 20 to 250 g/L in a neutral condition of pH 6.0 to 8.0, to suppress an immunoreaction so as to measure a subject substance without dilution; in Japanese Laid-Open Patent Publication No. Hei 10-332694, a method is disclosed in which the concentration of sodium chloride is set to 10 to 250 g/L in an acidic condition of pH 3.5 to 5.5 or in an alkaline condition of pH 9.0 to 12.0, to suppress an immunoreaction so as to measure a subject substance without dilution. Japanese Laid-Open Patent Publication No. Hei 11-344494 proposes, for example, the following method. Namely, in an immunological agglutination reaction in which the concentration of sodium chloride is set to 0.05 to 0.08 M in a neutral condition of pH 7.4 and an insoluble support particle is bound to an antibody or antigen as the one subjected to an immunoreaction, at least one dicarboxylic acid, selected from the group consisting of a malic acid, a glutaric acid, an adipic acid, a succinic acid, the salts and esters of these acids, is added to a reaction system in an amount of 1 to 20 wt %.
However, there has been a problem in that in any of the methods described in these publications, a measurement value of an immunoreaction is decreased in a measurement region other than a antigen excess region.
In view of the above-described conventional problems, accordingly, an object of the present invention is to provide an immunoreaction measurement method capable of easily increasing accuracy of measurement values, and an immunoreaction measurement reagent for use in the method. Another object of the present invention is to provide an immunoreaction measurement method capable of relaxing a limitation of a measurement range due to a zone phenomenon that occurs in an antigen excess region, and an immunoreaction measurement reagent for use in the method.